1. Field of the Invention
The present invention relates to an immunochromatoassay method, one of immunoassay methods, for detecting a target substance of a particular antigen or antibody using a specific reaction between antigen and antibody, and an immunochromatoassay kit used for the immunochromatoassay method.
2. Description of the Related Art
As one of immunoassay methods for detecting a particular antigen or antibody using a specific antigen-antibody reaction, a condensation method, in which a target substance in a test sample is caused to bind by immune reaction to an antibody or antigen sensitized on particles and the state of condensation of the particles caused by the binding is measured, is generally used because it is simple and allows, in particular, visual judgment.
In the immunoassay methods, competitive reaction and sandwich reaction are widely used. As one of the so-called sandwich reaction type assays, immunochromatography is known. For example, European Patent Publication No. 0582231 proposes a preferred form of the marker particle in the immunochromatography, and U.S. Pat. No. 5,266,497 proposes an apparatus and method for a solid phase assay.
In the immunochromatography, a target substance of antigen in a test sample is typically detected by the following steps: preparing a chromatograph medium having a test area by immobilizing particles sensitized by an antibody with respect to a target substance of antigen on a chromatograph medium (e.g., membrane of nitrocellulose) as solid phase particles or by directly immobilizing the antibody on the chromatograph medium, then preparing sensitized marker particles by sensitizing particles with an antibody which is specifically bindable to the target substance, and chromatographically moving the sensitized marker particles on the chromatograph medium with the test sample.
This results in that the immobilized antibody acts as an immobilized reagent and the sensitized marker particles specifically bind to the reagent in the test area of the chromatograph medium. Then, the presence or absence of a signal or the amount thereof generated when the sensitized marker particles are captured in the test area is visually judged, whereby the presence or absence of the target substance in the test sample or the amount thereof is measured. As for the particle for preparing the marker particle, a particle of colloidal metal, such as gold, platinum, copper, or ferric oxide, a particle of colloidal metal oxide, a particle of colloidal nonmetal substance, such as sulfur, or a dye particle is used.
As for the chromatograph medium, a membrane of organic macromolecule, such as nitrocellulose, having a comparatively high refractive index with a thickness of about 1 mm is generally used.
Generally, as shown in FIG. 4, the presence or absence of a signal (marker particles M) or the amount thereof is visually judged from above the thickness direction. In this case, a large difference in refractive index between membrane 102 (with refractive index=1.48) and solvent S (e.g., water, with refractive index=1.33) filled in the voids of the membrane causes the interface between them to be opaque due to light scattering. Consequently, even though the marker particles M are distributed over the entire region “d” in the thickness direction, only shaded marker particles M in region 106 adjacent to the surface (depth “ds” from the surface) are visually observed. If visual observation is performed after dropping an analyte solution and the solvent is dried, the refractive index difference between the membrane and air (refractive index=1) is further increased, causing more light scattering than when water is present in the voids and allowing only a shallower region to be visually recognized.
Where only a small number of antigens are present in the analyte solution, the number of antigens adsorbed adjacent to the surface is limited, so that an accurate visual judgment is difficult, thereby causing a problem that high detection sensitivity can not be obtained.
The present invention has been developed in view of the circumstances described above, and it is an object of the present invention to provide an immunochromatoassay method that allows an excellent visual judgment and highly sensitive detection. It is a further object of the present invention to provide an immunochromatoassay kit for implementing the immunochromatoassay method.